A polymerase chain reaction assay for simultaneous detection and quantitation of proto-oncogene and GAPD mRNAs in different cell growth rates.
A reverse transcriptase followed by a polymerase chain-reaction (RT-PCR) assay was developed for the simultaneous detection and quantitation of proto-oncogene (c-fos and c-myc) mRNAs using an internal standard mRNA glyceraldehyde-6-phosphate dehydrogenase (GAPD). Total cellular RNA was reverse transcribed and PCR amplified with oligonucleotide primers specific to GAPD and either c-fos or c-myc genes. In contrast to Northern blot analysis, the RT-PCR assay is rapid and sensitive enough to quantitate specific proto-oncogene levels from as little as 12-25 ng of total cellular RNA. The reliability of the assay was tested by measuring c-fos and c-myc expression in C3H 10T1/2 mouse embryo fibroblast cells under two different growth states: (a) quiescent cell entry into the proliferative cycle, and (b) plateau phase. Furthermore, the assay was used in measuring variations in c-fos or c-myc expression in HA-1 hamster cells following exposure to the cellular stressing agent, nitric oxide. In serum-stimulated cells, the RT-PCR measurements of transient increase in c-fos (16-fold at 30 min) and c-myc (10-fold at 1 h) mRNA levels were comparable to previously reported results in the literature using a Northern blotting assay. In addition, a two- to fivefold increase in c-fos mRNA levels was observed in plateau phase cells when compared to log phase growth. Furthermore, a transient increase in c-fos mRNA levels (threefold at 2 h) was also observed following cells' exposure to the stressing agent nitric oxide. These results suggest that the multiplex RT-PCR assay represents a significant improvement over current methods to quantitate specific cellular mRNAs under different growth conditions or following environmental insults.
Goswami
P C
PC
Section of Cancer Biology, Radiation Oncology Center, Washington University Medical School, St Louis, MO 63108, USA.
Albee
L D
LD
Spitz
D R
DR
Ridnour
L A
LA
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008809:Mice, Inbred C3H; D009569:Nitric Oxide / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000302:isolation & purification*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards
D016760:Proto-Oncogene Proteins c-fos; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases